In order to differentiate the extra virgin olive oils (EVOO) of different origin of purchase, such as monovarietal Italian EVOO with protected denomination of origin (PDO) and commercial-blended EVOO purchased in supermarkets, a number of samples was subjected to the analysis of volatile aroma compounds by both targeted gas chromatography/mass spectrometry (GC-MS) and untargeted profiling by comprehensive two-dimensional gas chromatography/mass spectrometry (GC×GC-TOF-MS), analysis of phenols by targeted high-performance liquid chromatography/mass spectrometry (HPLC-DAD-ESI/MS), and quantitative descriptive sensory analysis. Monovarietal PDO EVOOs were characterized by notably higher amounts of positive LOX-derived C6 and C5 volatile compounds, which corresponded to the higher intensities of all the assessed positive fruity and green odor sensory attributes. Commercial-blended EVOOs had larger quantities of generally undesirable esters, alcohols, acids, and aldehydes, which coincided with the occurrence of sensory defects in many samples. Many minor volatile compounds that were identified by GC×GC-TOF-MS were found to differentiate each of the two investigated groups. The differences between the groups with respect to phenols and taste characteristics were evident, but less pronounced. The results that were obtained in this study have undoubtedly confirmed the existence of the large heterogeneity of oils that are sold declared as EVOO. It was shown that GC-MS, GC×GC-TOF-MS, and HPLC-DAD-ESI/MS analyses have complementary outputs, and that their use in combination has advantages in supporting the results of sensory analysis and objectively differentiating these groups of EVOO. One of ive ones . The menive ones ,5,6, whiive ones .Oleaceae plants and they differentiate olive oil as unique among the other vegetable oils [p-HPEA-EDA or oleocanthal [Consumers, mostly because of its pleasant aroma, bitterness, and pungency, as well as its health effects, appreciate EVOO. The chemical parameters that are not regulated by the legislation, but they are certainly among the most meaningful for evaluating and understanding EVOO sensory and nutritional quality and they could serve as differentiators that are based on such criteria, are volatile aroma compounds and phenols ,8,9. Theble oils ,14. Secoble oils , althougble oils . EVOO puocanthal . EVOO phocanthal . In factIn this case study, two groups of olive oil, declared and sold on the Italian market as EVOO, were compared: i) monovarietal EVOOs with Protected Designation of Origin (PDO) directly purchased from producers and ii) commercial-blended EVOOs that were purchased from national grocery stores (supermarkets). Judging from the anecdotal experience, these two groups, which are frequently the subject of comparison and controversy among experts and consumers regarding their quality and price, should be fundamentally different, but this has not yet been scientifically confirmed to our knowledge. Accordingly, the aim of this study was to differentiate monovarietal PDO and commercial-blended EVOOs on the basis of volatile aroma and phenol composition, and to find reliable indicators of sensory quality among these compounds. It was considered that such findings would significantly contribute to EVOO diversification and they would help to clarify the interrelationship between EVOO origin, quality, and price, and in this way support the growth of the niche in the market segment of consumers informed and interested in healthy, quality products with remarkable diversity.Apart from being the first that aimed to differentiate EVOO on the basis of the origin of purchase, one of the most important novelties of this study is the utilization of a combined untargeted and targeted metabolomics approach utilizing powerful instrumentation and techniques, such as high throughput two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC×GC-TOF-MS) complemented by conventional monodimensional GC-MS for the analysis of volatile compounds, and high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) for the analysis of phenols. As a result, this study reported one of the most detailed and comprehensive qualitative and quantitative analytical characterizations of the volatile profile in EVOO up to date, with many compounds being identified (or tentatively identified) in EVOO for the first time. Additionally, it provided novel evidence regarding the diversity in sensory quality and volatile composition of olive oils sold declared as pertaining to the category of the highest quality grade (EVOO) and confirmed the need to re-evaluate the categorization criteria that were set by the current legislation.The results of sensory analysis are reported in a spider-web diagram in F-value, is shown in E)-2-hexanal, was also found in higher amounts in monovarietal PDO EVOOs, although it was not among the ones with the highest discriminative power judging from the F-values. However, (Z)-2-hexenal and (Z)-3-hexenal, carriers of major positive fruity and green notes, together with the isomers of 3-ethyl-1,5-octadiene and 4,8-dimethyl-1,3,7-nonatriene with unknown sensory relevance [The characteristic and unique flavor of EVOO, in particular its green and fruity attributes depend on many volatile compounds ,11. The elevance , turned elevance . In contelevance .The commercial-blended EVOOs from supermarkets were mostly characterized by the higher concentration of saturated esters, aldehydes, and alcohols . Such coE)-2-, (Z)-2-, and (Z)-3-hexenal, as well as 1-penten-3-one and unsaturated hydrocarbons, were characteristic for monovarietal PDO EVOO samples, and they could have contributed to generating positive green and fruity notes [PCA analysis clearly divided the samples in two groups . The majty notes  since a F-ratio.The first preliminary classification of monovarietal PDO and commercial-blended EVOO samples that were based on untargeted profiling information was obtained by applying PCA on raw data. However, not only selected bidimensional GC peaks with the highest potential for varietal differentiation, but also other peaks were tentatively identified on the basis of mass spectrum and linear retention index matching to improve its effectiveness and specificity and to obtain as much qualitative information as possible. F-values) for monovarietal PDO EVOOs were the minor ones, such as curcumene, octanal, 2-hexanol, ocimene, protoamenonine, valencene, etc. (Z)-2-hexenal, (E)-2-hexenal, 1-penten-3-one, and (Z)-2-penten-1-ol, are characteristic for this group of EVOO. The commercial-blended EVOO group was distinguished by a much larger number of volatile markers, including many of those often co-occurring with negative, defective sensory notes, such as 2-phenyl ethanol, isoamyl alcohol, isoamyl acetate, ethyl-2-methyl benzene, ethyl hexanoate, 3-methyl-2-buten-1-ol, 3-hydroxy-2-butanone, etc. [GC×GC-TOF-MS analysis extracted many minor compounds as statistically relevant for this study, which were not previously identified by GC-MS, either in this or in earlier studies. Such a result proved that the two techniques have complementary outputs and that their use in combination has serious advantages. Interestingly, several compounds that were most characteristic  was subjected to PCA , Perugia , Ragusa , Grosseto , Imperia , Brescia , Verona , and Riva del Garda . Furthermore, 25 commercial-blended EVOOs (price range from 3 to 12 €/L) were purchased from Italian grocery stores (supermarkets), which were selected according to Nielsen  data as among the most consumed during 2016 in Italy. All of the samples were stored in dark bottles at a controlled temperature of 15 °C before analysis, and gaseous N2 was added in the headspace to prevent oxidation each time that the bottles were opened.After preliminary selection from a larger group of high quality monovarietal EVOOs with PDO, samples that were produced from olives of Italian cultivars harvested in 2016 were collected from different geographical areas in Italy (price range from 20 to 30 €/L), including Reggio Calabria  and all aqueous solutions, including the HPLC mobile phase, were prepared with water purified while using a Milli-Q system . All of the analytical standards used for identification and calibration are listed in w/w in seeds oil). The headspace in the vial was equilibrated at 40 °C for 5 min. and the volatile aroma compounds were extracted at 40 °C for 30 min. The headspace was sampled using 2 cm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 μm fibre, purchased from Supelco . The volatile aroma compounds were desorbed in the GC inlet at 250 °C for 4 min. in splitless mode, and the fibre was reconditioned for 7 min. at 270 °C, prior to each analysis. Measurements were made while using a Thermo Trace GC Ultra gas chromatograph coupled to a Thermo Quantum XLS mass spectrometer Thermo Scientific , which was equipped with a PAL combi-xt  autosampler with a SPME option. A VF-wax capillary column  was used. The GC oven temperature gradient was starting from 40 °C for 4 min., 6 °C/min. up to 250 °C, and held for 5 min. Carrier gas was helium at the constant flow rate of 1.2 mL/min. The transfer line and the MS ion source were both set at 250 °C. Electron ionization was applied at 70 eV with an emission current of 50 mA. Mass spectra were recorded in centroid full scan mode at a scan time of 0.200 s from 30 to 350 m/z. Thermo Excalibur software  was used for all acquisition control and data processing. n-alkanes) were calculated and then compared to those from the literature. Semi-quantitative analysis was carried out and the concentrations of EVOO volatile aroma compounds were expressed as equivalents of the internal standard menthol in mg/kg of oil.Three grams of EVOO were put into a 20 mL glass headspace vial, and then spiked with 30 μL of internal standard solution  are reported in For GC×GC-TOF-MS analysis, a Gerstel MultiPurpose Sampler autosampler  with an agitator and SPME fiber was used to extract the volatiles from the EVOO sample vial headspace. The GC×GC system consisted of an Agilent 7890 A  that was equipped with a Pegasus IV time-of-flight mass spectrometer . A VF-Wax column (100% polyethylene glycol) 30 m × 0.25 mm × 0.25 μm  was used as a first-dimension (1D) column, and a RTX-200MS-column 1.50 m × 0.25 mm × 0.25 μm  was used as a second-dimension (2D) column. The GC system was equipped with a secondary column oven and a non-moving quadjet dual-stage thermal modulator. The injector/transfer line was maintained at 250 °C. The oven temperature program conditions were as follows: initial temperature of 40 °C for 4 min., programmed at 6 °C/min. up to 250 °C, where it remained for 5 min. The secondary oven was kept 5 °C above the primary oven throughout the chromatographic run. The modulator was offset by +15 °C in relation to the secondary oven; the modulation time was 7 s and 1.4 s of hot pulse duration. Such a modulation optimization was adapted for the analysis of minor volatiles. Helium (99.9995% purity) was used as carrier gas at a constant flow of 1.2 mL/min. The MS parameters included electron ionization at 70 eV with the ion source temperature at 230 °C, detector voltage of 1317 V, mass range of For GC×GC-TOF-MS data, LECO ChromaTOF Version 4.22 software was used for all acquisition control and data processing. Automated peak detection and spectral deconvolution with a baseline offset of 0.8 and signal-to-noise of 100 were used during data treatment. With these settings, it was possible to detect 1479 putative compounds. The identification of VOO volatile aroma compounds was performed by comparing the retention times and mass spectra with those of the pure standards, and with mass spectra from NIST 2.0, Wiley 8, and FFNSC 2  mass spectral libraries, with a minimum library similarity match factor of 750. Additional identification of volatiles was achieved by comparing the experimental linear temperature retention indices with those that were reported in the literature for 1D-GC. In total, 179 volatile aroma compounds were identified. To account for possible sample-to-sample variation, all of the intensities were normalized to the signal of menthol  and corrected for the mass added. The analyses were performed in triplicates, and the average values were used in further data elaboration.v/v) five times. Afterwards, 10 mL of hexane were added to the methanolic extracted solution, vortexed, and centrifuged for 5 min. at 5000 rpm. The methanol solution was collected and evaporated to dryness under vacuum. The extract was reconstituted with 2.5 mL of HPLC-grade methanol and then filtered through a 0.22 µm mPTFE filter before HPLC-DAD-MS analysis. Samples extraction was made according to . Five grv/v) with 0.1% formic acid (B) working in the gradient mode at a flow rate of 1 mL min−1. The solvent composition varied, as follows: 0 min., 30% B; 0–40 min., 60% B; 40–53 min. 95% B; 53.1–60 min. 30% B; then, the column was reconditioned. The column temperature was set at 35 °C and the injection volume was 10 µL [HPLC-DAD-ESI/MS studies were performed while using an Alliance 2695 HPLC with a diode–array detector DAD 2996) and a mass spectrometer detector qDa MS, Waters . The separation was achieved on a Synergi Polar reverse phase (RP)  analytical column from Phenomenex . The mobile phase for HPLC-DAD-ESI/MS analyzes was water with 0.1% formic acid (A) and methanol/isopropanol solution (90:10 96 and a p-HPEA-EDA (as tyrosol), p-HPEA-EA (as tyrosol), and acetoxypinoresinol (as pinoresinol).Phenols were quantified based on calibration curves of standards when available, while others were expressed as equivalents: 3,4-DHPEA-EDA-1 (as OH-tyrosol), 3,4-DHPEA-EDA-2 (as OH-tyrosol), 3,4-DHPEA-EA (as OH-tyrosol), Quantitative descriptive analysis of monovarietal and commercial EVOO samples was performed by the VOO sensory analysis panel comprised of eight assessors  that were trained for VOO sensory analysis according to the method that was proposed by IOC described in the European Commission Regulation . The panThe panel used a modified profile sheet that was expanded with particular positive odor and taste attributes . Single p < 0.05. These data, together with the results of sensory analysis (medians of intensities and grades), were further processed by principal component analysis (PCA) in order to better visualize the differences between the two groups of EVOO and explain them on the basis of the concentrations of volatiles and phenols. Three datasets  were separately treated. Prior to PCA analysis, the GC-MS and GC×GC-TOF-MS original datasets were reduced to only include the volatiles with the highest discriminative potency , combined with those regularly reported as among the most important for EVOO aroma. For the HPLC-DAD-ESI/MS dataset, first PCA was performed including all the identified phenols (p < 0.05), and those being close to that (0.05 < p < 0.10). Statistical data elaboration was performed by Statistica v. 13.2 software .GC-MS, GC×GC-TOF-MS, HPLC-DAD-ESI/MS, and sensory analyses data (concentrations of volatile aroma compounds and phenols) were subjected to one-way analysis of variance (ANOVA), and the average values were compared by Least Significant Difference (LSD) test at the level of  phenols , and theThe combined use of GC-MS and GC×GC-TOF-MS analysis for volatile aroma compounds proved to be a powerful analytical option, providing broad coverage of the volatilome, which is useful for the differentiation of the two classes of EVOO with respect to the origin of purchase: monovarietal PDO EVOO from family farms vs. commercial-blended EVOO from supermarkets, respectively. To our knowledge, this study provided one of the most detailed and comprehensive qualitative and quantitative analytical characterizations of the volatile profile in EVOO up to date, with many compounds being identified (or tentatively identified) in EVOO for the first time. Among them, many potential markers were extracted, despite the known (for PDO) and presumed  geographical and pedoclimatic heterogeneity and large variations in olive growing and oil producing parameters among EVOOs. Monovarietal PDO EVOOs were characterized by notably higher concentrations of desirable LOX-derived C6 and C5 volatiles, including the major ones that are known to be crucial contributors to the characteristic and appreciated EVOO green and fruity flavor. Such findings basically confirmed the results of the sensory analysis, which described the monovarietal PDO EVOOs by higher intensities and grades for positive sensory descriptors and attributes. On the other hand, the commercial-blended EVOOs had larger quantities of many volatiles that are known to originate from undesirable chemical and microbiological processes in olive and in olive oil, such as saturated esters, alcohols, acids, and aldehydes, which corresponded to the occurrence of sensory defects in many of the samples from this group. It is worth highlighting that a very large array of minor and unbeknown compounds, not reported or neglected in previous studies, was found to differentiate each of the two investigated EVOO groups, which point to the possibility that they also contributed to the perceived sensory notes. Targeted HPLC-DAD-ESI/MS profiling of phenols succeeded in differentiating monovarietal PDO from commercial-blended EVOOs to a much smaller extent, which was mostly due to the diversity of the concentration in monovarietal oils. Additionally, the differences that were observed during sensory analysis related to the corresponding taste attributes were not large. Nevertheless, the results of this study undoubtedly confirmed the large heterogeneity of oils, which are sold declared as EVOO in Italy, both in terms of chemical composition and sensory attributes, and in a way pointed to the possible reasons behind the existing large span of prices within this quality category.The approach that was proposed in this study is universal in nature and it could be applied for the characterization and differentiation of various other types of EVOO. The detailed profiles of volatile aroma compounds and phenols that can be obtained by the reported combination of powerful yet complementary techniques may serve experts, producers, and suppliers to better define typical sensory characteristics of EVOO in question, and in this way strengthen their identity and position on the market. From the technological point of view, understanding the compositional origins of the sensory typicity of particular EVOO might allow for more efficient quality management and control in production and more precise information to the consumers.


In vitro, H19 up-regulation promoted proliferation, migration, tube formation and vascular endothelial growth factor A (VEGFA) expression in HUVECs, and it was found to down-regulate microRNA-29c (miR-29c) expression. Bioinformatics analysis revealed that H19 mediated the above effects by binding directly to miR-29c. In addition, miR-29c expression was markedly reduced in vascularized corneas and its expression also decreased in bFGF-treated HUVECs in vitro. MiR-29c targeted the 3′ untranslated region (3′-UTR) of VEGFA and decreased its expression. These data suggest that H19 can enhance CNV progression by inhibiting miR-29c, which negatively regulates VEGFA. This novel regulatory axis may serve as a potential therapeutic target for CNV.Long non-coding RNA (lncRNA) H19 has been implicated in tumor angiogenesis. However, whether H19 regulates the progression of corneal neovascularization (CNV) is unclear. The present study aimed to determine the function of H19 in CNV and its possible molecular mechanism. Here, we found that the H19 levels were remarkably increased in vascularized corneas and basic fibroblast growth factor (bFGF)-treated human umbilical vein endothelial cells (HUVECs).  Corneal avascularity is an indispensable requirement for the cornea to remain transparent and for immune privilege. However, corneal neovascularization (CNV) can occur under conditions of infection, inflammation, immune response, chemical injury, trauma and impaired corneal innervation , leadingEmerging evidence suggests that non-coding RNAs play a major role in various biological processes, including normal development and disease progression. Non-coding RNAs are a class of RNA molecules without protein-coding potential that includes mainly short non-coding RNAs , piRNAs etc.) and long non-coding RNAs (lncRNAs). LncRNAs, which are over 200 nucleotides in length, can regulate gene expression in three main ways, including epigenetically, transcriptionally and post-transcriptionally. Notably, lncRNAs can function as endogenous ‘sponges’ that bind miRNAs to regulate miRNA expression and function . Among thttp://starbase.sysu.edu.cn/starbase2/index.php), there are potential binding sites between H19 and miR-29c. Thus, we speculated that H19 might promote CNV progression by suppressing miR-29c, which can target VEGFA. To the best of our knowledge, no published studies have explored the relationship between lncRNAs and miRNAs in CNV. Therefore, we explored the function of H19 and miR-29c in CNV progression by in vivo and in vitro experiments.MiRNAs, which are a group of short non-coding RNA molecules, can usually bind the 3′ untranslated region (3′-UTR) of a target gene. This action post-transcriptionally regulates the target gene through translational repression or mRNA degradation in a tissue-specific manner. Among all miRNAs, miR-29c has been found to suppress tumor angiogenesis by targeting VEGFA . In addiIn total, nine vascularized corneas were collected from patients diagnosed with CNV who underwent corneal transplantation at the Ophthalmology Department of the First Affiliated Hospital of Harbin Medical University between February 2018 and September 2018. Nine healthy corneal rims were obtained from the Eye Bank of Heilongjiang Province. The samples were immediately snap-frozen and stored at −80°C until RNA extraction. All patients and donors gave written informed consent prior to participating in the research. This research was carried out in accordance with the World Medical Association Declaration of Helsinki and was approved by the Harbin Medical University Research Ethics Committee .All animal experiments were conducted at the Animal Experimental Center of the First Affiliated Hospital of Harbin Medical University and were approved by the Harbin Medical University Animal Ethics Committee  in accordance with the guidelines of the Association for Research and the Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A total of 18 female Sprague–Dawley rats weighing 180–200 g were used for the animal experiments. The rats were divided randomly into three groups: a control group, a 7-day group, and a 14-day group. Each group consisted of six rats. A suture-induced rat CNV model was established as previously described . Briefly2 humidified incubator. The cells were cultivated with different concentrations of basic fibroblast growth factor  for the required time. In addition, pcDNA H19 was purchased from GenePharma . Human H19 siRNA (siH19) and the miR-29c mimic/inhibitor were provided by RiboBio . All cell transfections were performed according to the manufacturer’s protocol. Every experiment was repeated for three times independently.Human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) fetal bovine serum , 100 U/ml penicillin, and 100 µg/ml streptomycin  at 37°C in a 5% COC−ΔΔt method. Three independently repeated experiments were performed.Cells were collected, and total RNA was extracted using TRIzol reagent  according to the manufacturer’s protocol. The RNA concentration was determined by a Nanodrop Spectrophotometer . The Bulge-Loop™ miRNA qRT-PCR primer sets were designed and synthesized by RiboBio . Other primers were purchased from Invitrogen . The primer sequences are provided in the Supplementary Table S1. Bulge-Loop™ miRNA qRT-PCR Starter Kit  was used for detection of miRNAs via ABI 7500 Sequence Detection System . ReverTra Ace qPCR RT Kit  and SYBR-Green PCR Master Mix  were used for detection of H19 and VEGFA via ABI 7500 Sequence Detection System . The expression of miR-29 was normalized to that of U6 snRNA, while H19 and VEGFA were normalized to GAPDH. The relative expression level was calculated by using the 2Total protein was extracted from cells with RIPA lysis buffer  and quantitated with a BCA™ Protein Assay Kit . Then, 30 µg of protein was loaded into each well, and the samples were separated on 12% SDS/PAGE gels and transferred on to nitrocellulose membranes . Subsequently, the membranes were blocked with 5% non-fat milk  at room temperature for 1 h and incubated with the primary antibody at 4°C overnight. Then, the membranes were washed extensively and incubated with a fluorochrome-labeled secondary antibody  for 1 h at room temperature. Immunoreactivity was detected by an Odyssey fluorescent scanning system  and analyzed by Image Studio software. GAPDH was used as an internal control. Three independently repeated experiments were performed.HUVECs were seeded in 96-well culture plates at a density of 10000 cells/well after transfection and maintained for 24 h. The cell viability rate was assayed using a Cell Counting Kit-8 . CCK-8 solution was added to the medium and incubated at 37°C for 2 h. Optical density values were measured at 450 nm. Six replicate wells were established per group, and three independently repeated experiments were performed.HUVECs were seeded in six-well plates and cultured to 100% confluency after transfection. A sterile pipette tip was used to create a scratch in the monolayer perpendicularly across the center of the well. The floating cells were washed away with warm PBS three times. Images were captured 24 h after wounding under a microscope . Three independently repeated experiments were performed.The tube formation assay was performed as reported previously . Brieflyttest (two-tailed) and one-way ANOVA by using GraphPad Prism software . The data from each group are presented as the mean ± SEM. P-values <0.05 were considered statistically significant.Significant differences were determined by Student’s P<0.01). Then, an animal experiment was conducted to further verify this difference in expression. A suture-induced CNV model was chosen, as this is a classic model used to imitate the in vivo environment (P<0.05). However, there was no significant difference in the expression level between the 7-day group and the 14-day group. In addition, a cell experiment was performed to further explore the mechanism of non-coding RNAs in CNV progression. The PCR results showed that the expression level of H19 in the HUVECs progressively increased with increasing concentrations of bFGF; however, the difference was statistically significant at only the 20 ng/ml concentration (P<0.01). Therefore, we used the 20 ng/ml concentration for subsequent experiments. These findings imply that H19 may be strongly correlated with CNV progression.To determine the change in H19 expression during the CNV process, H19 expression in human cornea samples was first measured by real-time PCR. The results showed that the expression of H19 was higher in the CNV group than in the control group A; P<0.01ironment B. The PCironment C; P<0.05ntration D; P<0.01P<0.01), while H19 knockdown decreases these abilities (P<0.05). In addition, the results of the tube formation assay showed that the number of branches and the tube length in the pcDNA-H19 group were higher while those in the siH19 group were lower than those in the negative control (NC) group (P<0.05).Subsequently, we examined the potential functional role of H19 in HUVECs treated with bFGF. After confirming the transfection efficiency of H19 overexpression and knockdown A, the prC) group D; P<0.05P<0.001, B; P<0.05); aconcordantly, VEGFA increased with increasing concentrations of bFGF . Furthermore, we measured the expression of VEGFA after H19 overexpression or knockdown in HUVECs. As shown in A large body of evidence has documented that VEGFA plays an important role in the progression of CNV by contributing to the proliferation, migration and tube formation of HUVECs . TherefoP<0.05), while miR-29c expression increased when H19 was down-regulated (P<0.001).The above experiments indicated that H19 was positively correlated with VEGFA expression. In addition, lncRNAs are known to function as miRNA sponges to regulate mRNA expression and biological function. To elucidate the mechanism of this regulation, the potential complementary base pairs between lncRNA H19 and miRNAs were examined with bioinformatics software . MiR-29c may be a potential factor in the H19-VEGFA pathway, as it had binding sites for H19 A. Additiin vivo and in vitro. The expression of miR-29c was lower in the human CNV group than in the control group (P<0.001). In addition, the expression of miR-29c was markedly lower in the sutured corneas than in the control corneas (P<0.05). However, there was no significant difference in miR-29c expression between the 7-day group and the 14-day group. Furthermore, miR-29c expression in the HUVECs decreased after treatment with bFGF (P<0.01). These results indicate that miR-29c may be involved in CNV progression.Real-time PCR was also used to determine the expression of miR-29c during CNV progression  corneas B; P<0.05ith bFGF C; P<0.01Due to previous report that H19 can bind miR-29a/b in other disease models ,14,20,21P<0.05). Taken together, these results confirm that VEGFA is a downstream target of miR-29c.The potential role of miR-29c in CNV was assessed after miR-29c overexpression or knockdown. Real-time PCR was used to validate the efficiency of miR-29c mimic and inhibitor transfection A. The reP<0.001) and reversed by the miR-29c inhibitor (P<0.05). The VEGFA results were similar to the H19 results (To further examine the relationship between H19 and miR-29c and between miR-29c and its target VEGFA, HUVECs were cotransfected with siH19 and miR-29c mimics A,C/miR-2nhibitor B; P<0.05 results A–D. TakeIn the present study, we found for the first time that the lncRNA H19/miR-29c/VEGFA pathway is involved in corneal angiogenesis. The expression of H19 was increased in vascularized corneas and bFGF-treated HUVECs, and H19 could promote the expression of VEGFA by inhibiting miR-29c in HUVECs. This finding may be one of the underlying mechanisms in CNV progression.CNV is known to be caused by the decreased expression of anti-angiogenic factors and the increased expression of pro-angiogenic factors, including VEGF, bFGF, transforming growth factor-β, tumor necrosis factor-α, matrix metalloproteinases etc . Of thesLncRNA H19, which is a maternally expressed gene, is overexpressed in multiple types of cancer, including glioma, bladder cancer, gastric cancer, pancreatic cancer, and cervical cancer, and it plays a role in proliferation, immigration, invasion and angiogenesis ,22–25. IA number of studies have implicated miRNAs in the process of angiogenesis; for example miR-210 , miR-126in vitro. These results imply that H19 can promote VEGFA expression and angiogenic capability at least partially through the miR-29c/VEGFA axis.Emerging studies have shown that there is functional cross-talk between lncRNAs and miRNAs, but the role of their interaction in the progression of CNV remains unknown. Previous studies have shown that H19 can bind both miR-29a and miR-29b in other disease models ,14,20,21Although we know the potential role of H19 in the pathogenesis of CNV, we still have no idea what factors triggered the increase in H19 expression in CNV. Klein et al.  reportedSeveral studies have reported that the promotion or inhibition of non-coding RNAs helps to decrease CNV to some extent. Mulik et al.  and BhelThere were also other limitations to this research. Because it is difficult to collect human corneal tissues, it could be beneficial to verify our conclusions in studies involving more samples. In addition, we examined the changes in only sutured animal corneas; it may be necessary to examine the changes in other well-established CNV models, such as those involving cornea alkali burns. In addition, we chose an immortalized HUVEC lines instead of primary human corneal cells, which is another limitation. Thus, additional studies should be performed in the future.in vitro. The present study may aid in elucidating a potential therapeutic target for corneal angiogenesis in the future.In summary, the present research revealed for the first time that lncRNA H19 was expressed at high levels and that miR-29c was expressed at low levels in vascularized corneas. We also demonstrated that lncRNA H19 enhances angiogenic capability by acting as a molecular sponge of miR-29c to regulate the expression of VEGFA


Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging model for the study of biological asymmetry, biomineralization, and evolution and development. However, no systematic assessment of qPCR reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from several commonly studied tissues in L. stagnalis as well as across the entire body. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, beta-tubulin, ubiquitin, prenylated rab acceptor protein 1, and a voltage gated potassium channel. These genes exhibited a wide range of expression levels among tissues. The tissue-specific stability of each of the genes was consistent when measured by the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our data indicate that the most stable reference genes vary among the tissues that we examined . Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we provide suggestions for strong pairs of reference genes for single- and multi-tissue analyses of RT-qPCR data in L. stagnalis.Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression. To obtain reliable results with this method, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail ( Lymnaea stagnalis is a freshwater snail with an extensive history of proximate mechanism research. Previous studies have primarily focused on its neurobiology, as well as its endocrinology, immunology, and shell morphology , in situ hybridization, and transcriptomics (among others) have been established but have not yet been used extensively   (beta-actin (ACTB) (beta-tubulin (TUBB)  , glycera (GAPDH) , beta-acn (ACTB)  and betan (TUBB) . To estaLst-EF1α, Lst-GAPDH, Lst-ACTB, Lst-TUBB, and Lst-ubiquitin (UBI) as they are commonly employed reference genes. Messenger RNA transcripts encoding the prenylated rab acceptor protein 1 (Rapac1) and a voltage gated potassium channel (VGKC) were also assessed as analogs of these genes have recently been found to be stable in the terrestrial gastropod Cepaea nemoralis  where the RNA was washed and subjected to the on-column DNAse I treatment as per the manufacturer’s instructions. To confirm the effectiveness of the DNase I treatment, PCR was performed with primer sets for Lst-tyrosine hydroxylase  and Lst-EF1α with 20 ng of total RNA as a template as this reaction would not work in the absence of genomic DNA (gDNA) contamination. Lst-tyrosine hydroxylase was chosen as one of the gDNA contamination controls as it is frequently amplified by us in all of the tissues of interest as a part of a larger research project on neural development. No amplification was observed for either gene in any of the RNA samples after 40 cycles, thus the RNA samples were judged to be free of gDNA contamination.Total RNA was collected from each o 30 min . The aniTotal RNA was quantified in a QuBit 3.0 . Yields of 50–200 ng/µL dissolved in 30 µL of molecular grade water were common for roughly 40 mg of tissue mass. A sample of total RNA was measured with a spectrophotometer to confirm a 260:280 ratio of 1.8–2.0. Lastly, five µL of each RNA sample was denatured in five µL of 2× RNA Loading Dye  at 75 °C for 10 min and then immediately placed on ice. The RNA samples were loaded on a 1% agarose non-denaturing gel and run alongside a single stranded RNA ladder  to verify that the 28S and 18S rRNA bands were intact and the RNA samples were not degraded.Reverse transcription-PCR was performed with iScript Reverse Transcription Supermix for RT-qPCR  which contains a mixture of oligo(dT) primers and random hexamers. For RT-PCR reactions, 500 ng of RNA was added to four µL of iScript Supermix and topped up to 20 µL with molecular grade water as recommended by the manufacturer. RT-PCR took place in a Bio-Rad CFX Connect thermocycler . The RT-PCR program ran at 25 °C for 5 min, 46 °C for 20 min and 95 °C for 1 min.Candidate primers intended for qPCR were designed with CLC Main Workbench software . Primers were designed to have a length of 19–23 nucleotides, a melting temperature between 55 and 62 °C, a GC content between 40% and 60% and produce a product no more than 200 bp long. Additionally, the GC content of each primer was kept within 10% of its counterpart, and the melting temperature of each was kept within 3 °C of its counterpart.Two to six sets of primers were designed for each gene so that optimal primers could be selected for the qPCR reactions. The primers were tested on combined cDNA samples to minimize tissue-specific bias. Melt curves were performed to verify that one product was amplified. If the primers produced a single product, then five µL of the PCR reactions were run on a 2% (w/v) agarose gel at 60 V for roughly 30 min alongside a 50 bp DNA Ladder . The agarose gels were analyzed with a Bio-Rad ChemiDoc  to visualize the size of the product. Ultimately, one set of primers was selected for each candidate reference gene based on the quality of the PCR product. The primers for each candidate reference gene are listed in After the optimal primer sets were selected based on reaction specificity, primer efficiencies were calculated for each primer set. Primer efficiency curves were generated from RT-qPCR reactions on serial dilutions of RNA. The parent reaction contained 100 ng total RNA and four 1:5 dilutions were performed to generate five Cq values.2O. Each replicate of L. stagnalis tissue was subjected to qPCR reactions in triplicate. The qPCR reactions took place in a Bio-Rad CFX Connect thermocycler running a custom program. The custom qPCR program consisted of 95 °C for 30 s; 40 cycles of 95 °C for 15 s, 55 °C for 30 s. The plate was read by the machine to measure fluorescence at the end of each cycle.The minimum information for publication of quantitative real-time (MIQE) PCR experiments guidelines were followed throughout the collection of qPCR data . AmplifiM) values that are assigned following pair-wise variation measurements among genes. Genes with M < 1.5 are considered to be stable. The NormFinder algorithm compares intra-group variation  to inter-group variation  and assigns genes a stability value based on variation among Cq values. BestKeeper builds a stability index based on repeated pair-wise correlation analyses between every reference gene and judges reference gene stability based on the standard deviation (SD) from the index, genes that have a smaller SD will be ranked more highly by BestKeeper. Finally, RefFinder was used to combine the ranked results from each algorithm and assign each gene an overall rank.The expression stability of each gene was assessed with four computational algorithms: geNorm , NormFinL. stagnalis body. Given that a minimum of two reference genes are typically recommended for proper RT-qPCR data normalization, the recommended reference genes are generally expressed in terms of pairs.Data analyses were performed to compare the stability of among candidate reference genes in each tissue of interest. Following the tissue-specific analyses, we also combined all data for analysis together to simulate the conditions of a whole-body analysis. This approach allowed for the identification of which genes are most stable in each tissue of interest and provided evidence for which genes would be the strongest reference genes in an RT-qPCR experiment that used RNA from a whole GAPDH and Rapac1) to 106% (ACTB) with correlation coefficients (R2) of >0.980  are presented in L. stagnalis, the data from all tissues was combined and analyzed together with geNorm, NormFinder, BestKeeper, and RefFinder. The candidate reference genes spanned a wide range of expression levels when data from all tissue types was combined. Based on the Cq values, Lst-UBI was the most highly expressed gene (mean Cq 16.62 ± 0.09) and had the smallest overall Cq range whereas Lst-VGKC was the least expressed gene and had the highest biological variability as measured by the standard error of the mean  in six tissues of interest  and provided a separate analysis of all tissues combined. There were variable patterns of expression stability among the genes in the different tissues, but the tissue-specific rankings produced by the different algorithms were highly congruent. Lst-GAPDH and Lst-EF1α were the most suitable pair of reference genes in the CNS and penis. However, Lst-UBI also was one of the two most stable genes the tentacles, foot, and mantle when paired with Lst-GAPDH, Lst-ACTB, and Lst-VGKC, respectively. Finally, Lst-GAPDH and Lst-Rapac1 were the most stable in the lips. In the combined analysis, Lst-GAPDH, Lst-EF1α, and Lst-UBI were the most stable genes according to all algorithms. Thus, for future experiments using either whole animals or tissues or tissue combinations not included in our analysis, we recommend these three genes as the first candidates for validation as reference genes.We employed several established algorithms to analyze mRNA abundance data and identify stably expressed genes that are suitable as reference genes for RT-qPCR in EF1α appears to be the most effective reference gene in several mollusc species in addition to the CNS and penis of L. stagnalis  has generally produced positive results as a reference gene in several molluscan species (TUBA was unstable in C. nemoralis (Lst-TUBB to be highly unstable. ACTB has generally produced negative results as a candidate reference gene in molluscs (L. stagnalis are largely congruent with these previous findings, although Lst-ACTB was stable specifically in the foot. Finally, Rapac1 and VGKC were identified as suitable reference genes in C. nemoralis (Previous measurements of RT-qPCR reference gene stability has revealed some consistent patterns across the molluscs. tagnalis . In one t stress . Given te phylum . An analthe foot , similarbivalves  but was bivalves . Thus, G seasons . In our molluscs . Alpha-t species . Howeveremoralis  and we amolluscs  and the emoralis . These gLst-EF1α, Lst-GAPDH, and Lst-UBI were not the top ranked in every tissue as judged by RefFinder, they were relatively stable in all tissues tested which is reflected in the stability values provided by geNorm. Thus, we recommend these genes for use in multi-tissue comparisons in L. stagnalis. In particular, Lst-EF1α and Lst-GAPDH are a favorable pair as they have the most similar Cq ranges. Importantly, despite the demonstrated stability of these genes under baseline conditions reported here, it would be crucial to directly verify their stability under any experimental conditions (Despite the heterogeneity in top-ranked reference genes among the tissues, there was some consistency in expression stability across all tissues. Though nditions .L. stagnalis demonstrates that it is critical to select reference genes based on the tissues of interest. The results presented here should guide the selection of reference genes for tissue-specific RT-qPCR and thereby assist with future studies of gene expression in the snails.The validation of stable reference genes is necessary for the acquisition of reliable gene expression data. Therefore, it is important to perform a species-specific verification of reference gene stability before undertaking RT-qPCR experiments. The variable expression of the seven genes among the six tissue types we investigated in 10.7717/peerj.7888/supp-1Supplemental Information 1Genes are ranked in order from the least stable to the top two most stable.Click here for additional data file.10.7717/peerj.7888/supp-2Supplemental Information 2Genes are ranked in order from the least stable to the most stable.Click here for additional data file.10.7717/peerj.7888/supp-3Supplemental Information 3Genes are ranked in order from the least stable to the most stable.Click here for additional data file.10.7717/peerj.7888/supp-4Supplemental Information 4Reactions were performed in triplicate for each gene and each tissue. For this dataset, 500 ng of RNA was used in the reverse transcription reaction prior to qPCR.Click here for additional data file.10.7717/peerj.7888/supp-5Supplemental Information 5This is an additional dataset not referred to in the current version of the article. All raw Cq values for every qPCR reaction. Reactions were performed in triplicate for each gene and each tissue. For this dataset, 20 ng of RNA was used in the reverse transcription reaction prior to qPCR.Click here for additional data file.


Sleep-inducing PGD2 is produced in the brain by lipocalin-type PGD2 synthase (LPGDS). Three potential intracranial sources of LPGDS have been identified: oligodendrocytes, choroid plexus, and leptomeninges. We aimed at the identification of the site of synthesis of somnogenic PGD2 and therefore, generated a transgenic mouse line with the LPGDS gene amenable to conditional deletion using Cre recombinase (flox-LPGDS mouse). To identify the cell type responsible for producing somnogenic PGD2, we engineered animals lacking LPGDS expression specifically in oligodendrocytes (OD-LPGDS KO), choroid plexus (CP-LPGDS KO), or leptomeninges (LM-LPGDS KO). We measured prostaglandins and LPGDS concentrations together with PGD synthase activity in the brain of these mice. While the LPGDS amount and PGD synthase activity were drastically reduced in the OD- and LM-LPGDS KO mice, they were unchanged in the CP-LPGDS KO mice compared with control animals. We then recorded electroencephalograms, electromyograms, and locomotor activity to measure sleep in 10-week-old mice with specific knockdown of LPGDS in each of the three targets. Using selenium tetrachloride, a specific PGDS inhibitor, we demonstrated that sleep is inhibited in OD-LPGDS and CP-LPGDS KO mice, but not in the LM-LPGDS KO mice. We concluded that somnogenic PGD2 is produced primarily by the leptomeninges, and not by oligodendrocytes or choroid plexus.Injection of nanomolar amounts of prostaglandin D It is nsleep , a specific inhibitor of PGDS activity . All male mice  used in the present study were housed at a constant temperature (22 ± 1°C) with a relative humidity of 50 ± 2% on an automatically controlled 12:12 light/dark cycle (light on at 8:00 am). A mouse line called flox-LPGDS on a C57BL/6 background with a loxP-site-inserted LPGDS gene that is conditionally disrupted by expressing Cre recombinase was generated as previously described  and usedSeveral serotypes of AAV have been identified and are commonly used in neuroscience. These serotypes differ in their tropism (the types of cells they infect), making AAV a very useful system for preferentially targeting the gene of interest in specific cell types. We tested the ability for AAV to specifically infect the LM and the CP. We tested 5 different serotypes of AAVs  expressing the reporter protein mCHERRY in wild-type mice and discovered that the AAV of serotype 5 was the most efficient to target the CP, while only the serotype 8, when injected in postnatal mice (2-day-old), could infect the LM. Therefore LM-LPGDS KO mice were obtained by micro-injecting 6 μL of serotype 8 AAV-Cre into the lateral ventricle of 2-day-old flox-LPGDS mice. Briefly, neonatal mice were anesthetized by hypothermia on ice for 5 min before fixing to the pad of a stereotaxic arm. A glass micropipette with a 10- to 20-μm-diameter tip was introduced manually into the external corner of the right eye of the animal. Light pressure was applied to pass through the eye socket bone and deliver the AAV vectors into the lateral ventricle . Using aCP-LPGDS KO mice were obtained by micro-injecting serotype 5 AAV-Cre into the lateral ventricle of adult male flox-LPGDS mice. Briefly, mice weighing 24–28 g were anesthetized with pentobarbital , and 50 μl AAV5-Cre was stereotaxically microinjected into the left lateral ventricle  at a flow rate of 0.6 μL/min using a 100-μL Hamilton syringe and a syringe pump. Our observations indicate that the virus can disperse across ventricles and infect the remaining CP.OD-LPGDS KO mice were obtained by cross-breeding flox-LPGDS females with transgenic mice expressing Cre recombinase under control of the nestin promoter. The nestin promoter drives Cre expression only in neural precursor cells, leading to total KO of LPGDS in the derived cells, including the OD, but not in the LM or CP.n = 6 for each group).As controls, in the case of LM- and CP-LPGDS KO mice we used littermate Flox-LPGDS mice injected with AAVs of the same serotypes and administered following the same protocol, however, these AAVs express the fluorescent protein mCHERRY instead of Cre recombinase . In the case of OD-LPGDS KO mice, littermate control mice are born from the same parents but did not express Cre under the control of Nestin promoter  and EEG/EMG signals were recorded for 24 h. The EEG/EMG signals were amplified and filtered , then digitized at a sampling rate of 128 Hz, and recorded using SLEEPSIGN software  sequence from the AAV-hrGFP vector plasmid with the mCHE sequence. Subsequently, the gene coding for mCHE was replaced with the Cre recombinase coding sequence derived by polymerase chain reaction from the pBS185 plasmid (AAV-CMV-Cre and AAV-CMV-mCherry (mCHE) vectors from serotypes 5 and 8 were obtained as described earlier . Briefly plasmid . The serFollowing all procedures, the animals were deeply anesthetized with chloral hydrate  and perfused through the left ventricle of the heart with saline followed by neutral buffered 10% formalin. The brains were removed and placed in 20% sucrose in phosphate-buffered saline (PBS) overnight at 4°C for cryoprotection. The brains were then frozen on dry ice and sectioned at 30 μm on a freezing microtome. Immunohistochemistry was performed on free-floating sections with a primary antibody directed at LPGDS as described previously . Brieflyn = 6 for each group) in LM, CP, and OD on the brain PGDS enzymatic activity was measured by incubating 40 μg of brain extracts at 25°C for 1 min with [1-14C]PG H2  in 50 μL of 0.1 M Tris–HCl (pH 8.0) containing 1 mM dithiothreitol  as described previously  were harvested and immediately frozen in liquid nitrogen. They were then homogenized in ethanol containing 0.02% HCl at pH 2.0 and centrifuged at 500 g for 20 min. 3H-Labeled PGD2  was added to the supernatant as tracer to estimate recovery. The recovery value was approximately 60%. PGD2 was extracted with ethyl acetate, which was evaporated under nitrogen. The samples were then separated by HPLC  .The brains of LM-LPGDS KO, CP-LPGDS KO, and OD-LPGDS KO mice ( States) . PGD2 wat-test was used to analyze the amount of time spent in the different sleep-wake states. A one- or two-way ANOVA followed by the Fisher protected least significant difference test was used to determine whether differences in LPGDS, PGD2 contents, or PGDS activity in the brain of control or KO mice were statistically significant. In all cases, p < 0.05 was considered statistically significant.Data are presented as the mean ± standard error of the mean (SEM). For the sleep data analysis, an unpaired Student’s 2 that regulates sleep, we generated cell type-specific LPGDS KO mice for the CP, OD, and LM based on a mouse line in which the LPGDS gene is flanked by loxP sites in intron 1 and 6 and is amenable to conditional deletion by Cre recombinase  . To generate CP-LPGDS KO mice, we infused 50 μL of AAV-Cre, serotype 5 (AAV5-Cre), into the lateral ventricle of adult animals and 3 weeks later immunostaining confirmed the selective absence of LPGDS in the CP . Moreover, we crossed our floxed LPGDS mice with mice expressing Cre under control of the rat Nestin promoter and enhancer . In contrast, LPGDS immunostaining in the control flox-LPGDS animals revealed intense staining for LPGDS in the LM, CP, and OD .To determine which intracranial cell population produces the PGDgure S1) . To obtaenhancer . The Nesenhancer . This re2 in flox-LPGDS mice compared with that in control mice. Whereas the total amount of LPGDS in the brain of the CP-LPGDS KO mice remained unchanged compared with the control mice , the amount of LPGDS was reduced by 58.3 ± 11.1% (p < 0.001) and 75.7 ± 2.7% (p < 0.001), respectively, in the OD- and LM-LPGDS KO mice compared with the control mice. These results indicate that the contribution of the CP to the production of LPGDS in the mouse brain was negligible. In contrast, one-third to one-half of the brain LPGDS was produced by the OD.First, we measured LPGDS concentration in the brain of the LM-, CP-, and OD-LPGDS KO mice. We then determined the amount of PGDp = 0.6082), whereas it was significantly reduced by 39.7 ± 6.5% (p = 0.0028) and 78.9 ± 6.9% (p = 0.0004), respectively, in the OD- and LM-LPGDS KO mice . These findings are in good agreement with the distribution of LPGDS in the brain of the LM-, CP-, and OD-LPGDS KO mice.We next measured PGDS enzymatic activity of the LM-, CP-, OD-LPGDS KO mice and their respective control littermates. PGDS activity in the brain of the CP-LPGDS KO mice remained unchanged compared with the control animals  and 55.5 ± 1.8% (p = 0.001), respectively, compared with the control flox-LPGDS animals . On the other hand, no change in PGD2 concentration was observed in the brain of CP-LPGDS KO mice compared with the control mice (p = 0.9078).Finally, we measured the total amount of PGD4 at a dose of 5 mg/kg at 10:00 am when mice are mostly asleep, as previously described. SeCl4 is a specific inhibitor of PGDS activity that can inhibit natural sleep in wild-type and hematopoietic PGDS KO mice, but not LPGDS KO mice . Furthermore, we analyzed EEG/EMG recordings obtained during a 4-h period after i.p. injections of SeCl4 (5 mg/kg) and vehicle. The total amount of time spent in sleep was reduced in the CP- and OD-LPGDS KO mice as well as in the control mice during the 3-h period following the injection of SeCl4 compared with vehicle, but in the LM-LPGDS KO mice, the time spent in sleep after the injection of SeCl4 was indistinguishable from that following the vehicle injection .We analyzed the sleep-wake pattern of the LM-, CP-, and OD-LPGDS KO mice by measuring their EEG and EMG activity before and after an i.p. injection of the PGDS inhibitor SeCl KO mice . The bas4 is shown in Figure 3B. The total amount of sleep was drastically reduced in the CP- and OD-LPGDS KO mice during the 4 h following the SeCl4 injection  compared with the vehicle control . The sleep amount was similarly reduced by the injection of SeCl4  in the control animals (flox-LPGDS). By contrast, no sleep reduction was observed in the LM-LPGDS KO mice, indicating their complete insensitivity to the SeCl4 injection . These results suggest that LPGDS expressed in the LM, but not the CP or OD, is involved in the regulation of physiologic sleep.The changes in the time-course of sleep in the cell type-specific CP-, OD-, and LM-LPGDS KO mice during the 4 h following the i.p. injection of 5 mg/kg SeCl4) or PGD2 receptor, subtype DP1, antagonist (ONO-4127Na) inhibits sleep in rats and mice, indicating that the PGD2 system is crucial for maintaining physiologic sleep (2 responsible for the induction of sleep, however, has been debated over the last 30 years (2, which is secreted into the CSF, where it stimulates DP1 receptors as a sleep hormone. In the same pathway, adenosine is then released as a secondary sleep-promoting messenger and activates adenosine A2A receptor-expressing neurons (1 and A2A receptors are expressed, however, remains to be clarified.The administration of an LPGDS inhibitor (SeClic sleep . The spe30 years ,b. Using neurons . Through neurons . Where DThe basal sleep behavior of the cell type-specific KO mice for the CP and OD was identical to that of their control littermates, while the LM-LPGDS KO mice exhibited slightly reduced REM sleep activity during the daytime. The absence of a major sleep phenotype in the conditional LPGDS KO mice is in good agreement with previous reports of global LPGDS KO mice . The moslpgds gene deleted. Indeed, other cells such as tanycytes -a special ependymal cells found in the third ventricle of the brain and on the floor of the fourth ventricle- or ependymocytes -a type of glial cell forming the ependyma, a thin neuroepithelial lining the ventricular system- were most likely in contact with the AAVs and might have been infected. However, it is unclear if the serotypes of AAVs used in this study can effectively infect such ependymal cells. Furthermore, even if these and other cell types have also been infected, then started to express Cre recombinase, and were deleted of their lpgds gene, we believe that the effect would have remained unnoticed. To our knowledge, these cells have never been reported to express LPGDS and therefore animals should not be impacted by the potential ectopic expression of Cre recombinase.While using AAVs expressing Cre recombinase permitted to delete the expression of LPGDS in the CP and the LM, we cannot exclude the possibility that other cell types might also have been infected and their copy of 2 that is involved in the regulation of physiologic sleep. Our findings in cell type-specific LPGDS KO mice demonstrate that the somnogenic PGD2 is primarily produced by LPGDS in the LM.Lipocalin-type PGDS is implicated in the production of PGDThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.YC and YU designed the research and wrote the paper. YC and KA performed the research. YO contributed the transgenic mice. YC, KA, MKK, and MK analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PD-L1) mRNA in salivary exosomes from patients with periodontitis and non-periodontitis controls. The purposes of this study were to establish a procedure for isolation and detection of mRNA in exosomes from saliva of periodontitis patients, to characterize the level of salivary exosomal PD-L1, and to illustrate its clinical relevance. Bioinformatics analysis suggested that periodontitis was associated with an inflammation gene expression signature, that PD-L1 expression positively correlated with inflammation in periodontitis based on gene set enrichment analysis (GSEA) and that PD-L1 expression was remarkably elevated in periodontitis patients versus control subjects. Exosomal RNAs were successfully isolated from saliva of 61 patients and 30 controls and were subjected to qRT-PCR. Levels of PD-L1 mRNA in salivary exosomes were higher in periodontitis patients than controls (P < 0.01). Salivary exosomal PD-L1 mRNA showed significant difference between the stages of periodontitis. In summary, the protocols for isolating and detecting exosomal RNA from saliva of periodontitis patients were, for the first time, characterized. The current study suggests that assay of exosomes-based PD-L1 mRNA in saliva has potential to distinguish periodontitis from the healthy, and the levels correlate with the severity/stage of periodontitis.Periodontitis is the most prevalent inflammatory disease of the periodontium, and is related to oral and systemic health. Exosomes are emerging as non-invasive biomarker for liquid biopsy. We here evaluated the levels of programmed death-ligand 1 ( Periodontitis is one of the most prevalent disease in dentistry, impairing the integrity of the periodontium and leading ultimately to tooth loss. Periodontitis is a chronic inflammatory disease caused by microorganisms colonizing the dentogingival interface . CurrentGiven the close relevance of inflammation to periodontitis, immune-regulatory factors have been explored as biomarker of periodontitis . ProgramPorphyromonas gingivalis  is a keystone pathogen in chronic periodontitis  membrane-encapsulated vesicles containing nuclei acids and protein cargo, and secreted by eukaryotic cells into the circulation . The condontitis , it is uThis study is a prospective observational investigation at the First Affiliated Hospital of Shantou University. From June, 2017 to June, 2018, 61 periodontitis patients and 30 control subjects were enrolled in the study with informed consent. Each recruited subject was inquired in detail about the medical history and accepted a thorough periodontal examination. Diagnosis of periodontitis was based on assessment of probing pocket depth (PD) and clinical attachment loss (CAL). Patients who fulfilled the requirement of generalized chronic periodontitis according to the classification by the American Academy of Periodontology in 2007  were recFor each individual, PD, CAL and bleeding on probing (BP) values were measured with a periodontal probe. A single calibrated examiner assessed these clinical parameters. Control individuals (control group) did not present any sign or symptom compatible with periodontal disease . On the other hand, the periodontitis group consisted of patients with mild, moderate or severe periodontitis   with at Human gingivae were obtained from all periodontitis patients. Gingiva was harvested during tooth extractions for periodontal reasons in the Department of Stomatology of First Affiliated Hospital of Shantou University. Written informed consent and approval of the Ethics Committee of the SUMC were obtained.g for 15 min at 4°C to remove cells and cellular fragments. As much supernatant as possible was collected into new 1.5-mL tubes and stored at -80°C.Saliva was collected in the morning (8 am to 10 am) from all subjects. During the collection period, subjects were instructed to refrain from eating, drinking or using oral hygiene products for at least 1 h prior to collection, and received no stimulation of salivation. After rinsing the mouth with water, each subject spat 3–5 mL saliva into a 35-mm dish. Subjects were reminded not to cough up mucus, and saliva was collected within 30 min from spitting. These saliva samples were pipetted into 1.5-mL tubes and were kept on ice during processing which did not exceed 60 min. The samples were then centrifuged at 3,000 ×1) was performed to examine the association between gene sets and gene expression  vs. unaffected gingival tissue (n = 69) from GSE16134 was ordered from high to low. We performed GSEA analysis to examine the correlation between periodontitis and the PD-L1 signature pathway, followed by the protocol available at the GSEA website1.Gene set enrichment analysis . Briefly, ExoQuick-TCTM solution was added to saliva at a 63/250 ratio and mixed by inverting the tubes several times. Exosomes were precipitated by refrigeration at 4°C overnight, and then collected by centrifugation twice at 1,500 ×g for 30 and 5 min, respectively, in order to remove the supernatant. Supernatant was discarded, and the pellet resuspended in 300 μL TRIZOL for RNA isolation or 20 μL PBS for protein isolation.ExoQuickTotal RNA was extracted from exosomes and cell lysates using TRIzol reagent as per the manufacturer’s protocol (ZYMO RESEARCH). RNA was eluted with 25 μL of RNAse-free water. RT-qPCR was performed using an Absolute Blue QPCR SYBR Green Low ROX mix (Thermo Scientific) on an Applied Biosystems’ 7500 real-time PCR system. The Rn value  was the fluorescent signal from SYBR Green normalized to the signal of the passive reference dye for a given reaction. No-template and no-RT reactions were performed as negative controls. All assays were performed in 3 separate RTs followed by triplicate qPCR, and the results are shown as the average fold change relative to GAPDH which served as an internal control. Primers for RT-qPCR were:GAPDH F: 5′-TGCACCACCAACTGCTTAGC-3′GAPDH R: 5′-GGCATGGACTGTGGTCATGAG-3′PD-L1 F: 5′-TGCCGACTACAAGCGAATTACTG -3′PD-L1 R: 5′-CTGCTTGTCCAGATGACTTCGG-3′.Exosomal pellets were resuspended in PBS and re-pelleted by centrifugation and then extracted with RIPA buffer (Santa Cruz Biotechnology). Total protein lysates were prepared and analyzed by immunoblotting using anti-ALIX , anti-TSG101 , anti-CD63 , anti-CD9 , anti-CD81 , anti-Calnexin , anti-LC3 , anti-NLRP3  and anti-NLRP4  as described previously .g for 70 min to re-pellet the exosomes. The exosomes pellets were resuspended in 30 μL PBS, and a 10 μL aliquot of the suspension loaded onto formvar carbon-coated grids and allow to stand for 5 min at room temperature. Next, the exosomes were fixed in 2% paraformaldehyde for 5 min at room temperature and washed thrice with PBS. Excess liquid was drained by gently touching the edge of the grid with clean filter paper. The grid was slightly touched onto a drop of 2% uranyl acetate for 1 min and embedded in a mixture of uranyl acetate (0.8%) and methyl cellulose (0.13%). Excess liquid was drained off, and then the grid was allowed to air dry for several minutes prior to examination under a transmission electron microscope   .RRID: SCR_014239, version 2.3). All measurements were recorded at room temperature.Total particles in human salivary samples were analyzed by nanoparticle tracking by the A & P Instrument Co., Ltd. , using a NanoSight LM10 system . Each sample was diluted in nanoparticle-free PBS and analyzed three times. Data was collected and analyzed using the nanoparticle tracking analysis (NTA) software  or RPMI-1640 medium  supplemented with 10% heat-inactivated fetal bovine serum , 10 mmol/L glutamine, 100 units/ml penicillin , and 100 μg/ml streptomycin (Sigma). TE1 cells were provided by Dr. X.C. Xu . For LC3 detection, TE1 cells were collected after starvation  for 6 h.t-test, Pearson correlation and chi-square test.All statistical analyses were performed using the SPSS 19.0 statistical software package  and Prism V6.01 (GraphPad). Summary statistics reporting means and standard errors were stated as appropriate. Statistical methods used included Given the association of periodontitis with inflammation, and the importance of PD-L1 in inflammatory immunity, we analyzed database to test whether periodontitis correlates with PD-L1. GSEA analysis revealed that periodontitis is positively associated with an inflammation signature, and PD-L1 is positively correlated with an inflammation signature in periodontitis . AnalysiThis pilot study was conducted at Shantou University Medical College. Sixty-one periodontitis patients and 30 control subjects were enrolled in the study . With anIsolation of exosomes from human saliva was confirmed by transmission electron microscopy (TEM)   and nanoPD-L1 mRNA expression, in 61 periodontitis patients and 30 control subjects, by qPCR analysis. As shown in PD-L1 expression compared with the mean salivary exosomal PD-L1 expression in control subjects. Mean salivary exosomal PD-L1 expression in the periodontitis patients was found to be about 10-fold higher when compared with the paired control subjects (P< 0.001). Similarly, mean gingival PD-L1 mRNA expression in the periodontitis patients was more than 10-fold higher than that in control subjects (P< 0.001). More importantly, salivary exosomal PD-L1 mRNA levels highly correlated with gingival PD-L1 mRNA levels in periodontitis patients  was chosen to classify patients into high-(n = 31) and low-PD-L1 (n = 30) groups  groups . High PDrameters . Our resPD-L1 mRNA expression in exosomes derived from saliva of periodontitis patients, and have evaluated the clinical relevance of the levels of salivary exosomes PD-L1 mRNA in the disease. One of the main findings was that the level of salivary exosomes PD-L1 mRNA in periodontitis patients is highly distinct relative to non-periodontitis controls. Moreover, high level of salivary exosomes PD-L1 was associated with an advanced stage of periodontitis, suggesting it can reflect disease progression. This is the first to establish a procedure of detection of saliva-based exosomal PD-L1 in disease, and the first salivary exosomal biomarker for periodontitis. In the majority of previous investigations of periodontitis biomarker, the specimens are either gingival tissues or gingival crevicular fluids (GCF)  . Howeverds (GCF) . To our + T lymphocytes and CD8+ T lymphocyte of chronic periodontitis patients was upregulated (PD-L1 mRNA in saliva has potential to distinguish periodontitis from the healthy, and the levels correlate with the severity/stage of periodontitis. The increased expression of PD-L1 in severe periodontitis might due to the requirement of the body for more PD-L1 to inhibit destruction of inflammatory tissues. It has been shown that tumor cells upregulate PD-L1 expression to evade host immune response and thereby to maintain disease (PD-L1 has been reported to play an important role in a wide range of cancers and inflammation-originated diseases including periodontitis . The rolegulated . High exegulated . The pre disease . Moreove disease . PD-L1 i disease , ovarian disease , lung ca disease , and upr disease . Our ana disease , there a disease . We here disease , the proPD-L1 in saliva was enriched in periodontitis and was associated with advanced stage of disease. These findings are worthy to validate in further investigations with expanding samples.In summary, we described the procedure for isolating mRNA from exosomes derived from saliva of periodontitis patients. Our further assay showed that exosomal in vitro experiments about patient specimen analysis, bioinformatics assay, and analyzed data. JY, YL, XX, S-CY, and HZ interpreted data and wrote the manuscript. JY, S-CY, DY, and HZ contributed to discussion and reviewed the manuscript.HZ conceived and designed the experiments. JY and DY provided patients and the study materials. YL, KL, XX, ZY, HD, and ZJ performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Similarly, among dual users, nicotine dependence levels were higher when using an e-cigarette (mean 4.7) compared to using traditional cigarettes . Habits and behaviors associated with the use of e-cigarettes did not differ significantly (p > 0.05) between exclusive e-cigarette users and dual users. The findings suggest that e-cigarettes may have a higher addictive potential than smoked cigarettes among young adults.E-cigarettes are often considered less addictive than traditional cigarettes. This study aimed to assess patterns of e-cigarette use and to compare nicotine dependence among cigarette and e-cigarette users in a group of highly educated young adults. From 3002 healthy adults, a representative group of 30 cigarette smokers, 30 exclusive e-cigarette users, and 30 dual users were recruited. A 25-item questionnaire was used to collect information related to the patterns and attitudes towards the use of cigarettes and e-cigarettes. The Fagerström test for nicotine dependence (FTND) and its adapted version for e-cigarettes were used to analyze nicotine dependence in each of the groups. The nicotine dependence levels measured with FTND were over two times higher among e-cigarette users (mean 3.5) compared to traditional tobacco smokers (mean 1.6;  Nicotine is a highly addictive substance and adolescents and young adults may be at high risk of addiction ,2. NicotElectronic cigarettes (e-cigarettes) are a relatively new form of nicotine delivery device and the prevalence of their use is rapidly growing in many countries ,8,9. E-cDue to uncertainty about the dependency potential of e-cigarettes, we decided to carry out a study aimed to: (1) assess the patterns of e-cigarette use by exclusive e-cigarette users and dual users and (2) compare nicotine dependence levels among young adults who use cigarettes or e-cigarettes. We chose to examine the problem in a highly educated group of university students under the assumption that their perception is less biased by commonly held beliefs and stereotypes of e-smoking safety and addiction mechanisms.This study is a continuation of a survey-based and multicentered international project, the YoUng People E-Smoking Study (YUPESS) . From thn = 93) were invited to participate in the second part of the study. Exclusion criteria included occurrence of any chronic diseases, history of lung diseases , presence of any allergic diseases, any medication intake (current or in the last 2 weeks), acute illnesses or infections in the last 2 weeks, influenza vaccination in the last 2 weeks, and current pregnancy or lactation. Among exclusive e-cigarette users, three met the exclusion criteria and six refused. Finally, 30 exclusive e-cigarette users were included. Among dual users, four met the exclusion criteria. To keep an equal number of participants per group, out of 50 eligible dual users, we randomly selected a group of 30 dual users, using randomization procedures available in the Statistica package. An additional group of 30 people was drawn randomly from a group of cigarette smokers (n = 433).A cross-sectional survey was performed between January and March 2018. Three fields of study  were randomly selected from a group of 245 different fields of study within Universities in Katowice. The questionnaire was addressed to all the students within each selected field of study. Data were collected from 3002 participants. Out of all the participants, we identified 39 exclusive e-cigarette users and 54 dual users. All subjects who used e-cigarettes . Statistical significance of differences between continuous variables was analyzed using an independent samples t-test or a Mann-Whitney U test where appropriate. Statistical testing to compare categorical variables was completed using an independent samples chi-square test. We assessed strength of association by calculating odds ratios (OR). We used 95% confidence intervals (CI) and defined statistical significance as p = 0.03). Duration of e-cigarette use was comparable between exclusive e-cigarette users and dual users being 29.0 ± 24.1 and 27.7 ± 17.4 months, respectively (p = 0.6). There were no statistically significant differences in sex, age, or the daily number of cigarettes or e-cigarette smoking sessions between groups (p > 0.05).Of the adults included , the mean smoking duration was 50.0 ± 32.0 months among smokers and 67.3 ± 30.5 months among dual users (p > 0.05) between e-cigarette users and dual users. Users of e-cigarettes (n = 60) consumed an average of 4.2 mL of e-liquid per day, with the most frequently chosen e-liquid being that containing 6 mg of nicotine in 1 mL of e-liquid. None of the subjects used nicotine free e-liquids. The study population was dominated by individuals who prepared e-liquids themselves: 66.7% of e-cigarette users and 74.1% of dual users (p = 0.9) had chosen devices that allow technical modifications of the e-cigarette such as voltage, power, and resistance of the heater.Habits and behaviors associated with the use of e-cigarettes, including nicotine content in the e-liquid, e-liquid consumption, type of e-liquid used, and the number of e-cigarettes used, did not differ significantly . The most common reasons stated for starting to use e-cigarettes were it being less harmful to health, price, and desire to quit traditional smoking. The reasons stated for using e-cigarettes are presented in Almost all exclusive e-cigarette users (96%) had ever tried cigarette smoking. Out of the 60 e-cigarette users, 18.5% initiated nicotine use by e-cigarette. E-cigarette, as a first nicotine product, was declared by 16% of exclusive e-cigarette users and 20.7% of dual users  indicated that traditional cigarette smoking offers more smoking satisfaction (46.2%) compared to using e-cigarettes (23%). Also, smoking a cigarette in a group of friends, in combination with a cup of coffee, after a meal, or after sex was associated with a greater subjective feeling of satisfaction compared to the feelings associated with the use of an e-cigarette in the same situations. Dual users indicated that the smell (92.9%) and the taste (82.1%) of e-cigarettes as well as the subjective feeling associated with the inhalation and exhalation of vapor generated from e-cigarettes were the primary advantages of e-cigarettes over traditional cigarettes.p = 0.002; p = 0.03) among dual users. E-cigarette users were more likely to use an e-cigarette in the first 30 min after waking and were more likely to find it difficult to refrain from using e-cigarettes in places where it is forbidden (The average FTND score among exclusive e-cigarette users was over twice as high (mean 3.5 versus 1.6) as among traditional cigarette smokers  or financial reasons. The participants consumed relatively large amounts of nicotine-containing e-liquid . More than half of the participants chose technically advanced e-cigarette devices.p < 0.001). Similarly, among dual users, nicotine dependence levels were higher when using an e-cigarette (mean 4.7) compared to a traditional cigarette . The high levels of nicotine dependence observed among young adult e-cigarette users compared to smokers in our study suggest this group may be at high risk of addiction.Our key finding was that nicotine dependence levels measured with FTND were over two times higher among exclusive e-cigarette users (mean 3.5) compared to traditional tobacco smokers  compared to dual users, although this was not statistically significant.p = 0.03) [Our findings contrast with results obtained by González et al. , Etter e = 0.03) . Etter e = 0.03) , althoug = 0.03) . When co = 0.03) . Also, p = 0.03) .Several hypotheses should be considered to explain why we found higher levels of addiction among e-cigarettes users compared to smokers in contrast to findings in studies by González et al. 2017) , Etter e017 , EttIn our study, e-cigarette users consumed an average of 4.2 mL of e-liquid per day. Slightly lower results were obtained by Hajek et al. at 3.3 mL per day  and DawkStrengths of this study include the detailed description of patterns of use of young, experienced e-cigarette users and the investigation of the degree of addiction of e-cigarettes and cigarettes using an established measure of addiction among a key population group—young adults. Further separation of this group into groups of exclusive e-cigarette users and dual users allowed for a fuller understanding of the differences in behaviors and addiction presences between these groups.There are several potential limitations of the current study. First, our study was limited to a small sample of young e-cigarette users from one region of Poland. This is a specific group of e-cigarette users which was identified among the 3002 young adults who participated in an across-sectional survey (YUPESS—Katowice branch), with the sample being representative of young adults with higher education (university students) in Poland  but not E-cigarettes are promoted as a safer alternative to traditional cigarettes. This view is also shared by e-cigarette users ,36. ExclIn conclusion, in this work the use of e-cigarettes among young adults was shown to result in higher nicotine dependence levels than nicotine dependence related to tobacco cigarette use. In addition, a stronger dependence on the e-cigarette compared to the traditional cigarette identified in dual users indicates that e-cigarettes may be highly addictive. Further research is necessary to provide evidence on the role of e-cigarettes in smoking cessation, especially in young adults. Social normalization of smoking behaviors by allowing e-cigarette use should be monitored.


However, it is not clear how long this improved epithelium sealing can be maintained. We subsequently investigated whether the positive effect of Ca-HT to promote sealing between the PIE and implant was sustained longer term. Maxillary molars were extracted from rats and replaced with either Ca-HT implants (Ca-HT group), distilled water-HT implants (DW-HT group) or non-treated implants (control group). After 16 weeks, the majority of implants in the Ca-HT group remained at the maxillary with no apical extension of the PIE. Conversely, half the number of control implants was lost following down-growth of the PIE. The effect of Ca-HT on migration and proliferation of rat oral epithelial cells (OECs) was also investigated. In OECs cultured on Ca-HT Ti plates, protein expression in relation to cell migration decreased, and proliferation was higher than other groups. Surface analysis indicated HT enhanced the formation of surface TiO2 layer without altering surface topography. Consequently, Ca-HT of Ti reduced PIE down-growth via tight epithelial attachment to the surface, which may enhance implant capability for a longer time post-implantation.We previously reported that CaCl Dental implants are reported to have high success rate but peri-implantitis is one of the most important reason for the failure of implants. Peri-implantitis is initiated by the deterioration of implant-gingival adhesion associated with inflammatory cells , with thIn particular, epithelial down-growth around dental implants destabilizes the soft tissue-implant interface by providing a route for invasion of external pathogens, which may be a reason for implant failure. Periodontal pocket formation provides a moist, warm and anaerobic environment for bacterial colonization, with mechanical cleaning difficulties. Although the epithelial tissue can go down through damaged connective tissue, healthy collagenous matrix can stop this migration . Down-grIn our previous study using rodent, both the integration of soft tissue cells with titanium and the epithelial sealing against foreign body penetration were improved at 4 weeks after implantation when the titanium implant was hydrothermally-treated with calcium chloride (Ca-HT) . HoweverCommercially available, mirror-polished commercially grade 1 pure titanium plates ) and experimental implant bodies  received a hydrothermal treatment with 10 mmol/L CaCl2 at 200 °C in a pressure pot for 24 h [Implants in the control group (Cont) received no treatment. Implants in the HT with distilled water (DW) group (DW-HT) received a hydrothermal treatment in DW at 200 °C in a pressure pot for 24 h. Implants in the CaClfor 24 h ,14. AfteThe composition of each specimen was determined using an X-ray diffractometer  operated at 40 kV and 30 mA. The diffraction angle was continuously scanned from 20°–90° in 2 theta.Rats  were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, after approval of the research protocol by the ethical committee of Kyushu University . The Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for the execution and submission of studies in animals were followed. Details of the implant placement were described in the previous manuscripts . In brieHorseradish peroxidase  was applied around the implant at 16 weeks after implantation to evaluate the secureness of epithelial sealing. The procedure for topical application of HRP was similar to that reported previously . Under sTissues were prepared as described previously . After 12O2 in 0.05 M Tris-HCl buffer, pH 7.6, for 5 min at room temperature. For controls, additional sections were incubated in DAB without H2O2. All sections were observed with a light microscope [Several 10-μm-thick sections were washed with 0.01M PBS and incubated in 3,3’-Diaminobenzidine tetrahydrochloride  medium containing 0.02% DAB and 0.006% Hcroscope .2O2, and counterstained with hematoxylin.As described previously, an avidin-biotin complex (ABC) procedure was employed for immunohistochemical staining . In brieFive frozen sections were selected from the buccal side of peri-implant epithelium (PIE) and used for the linear measurements, with the landmarks to measure the vertical distance between the top of the gingival edge and bottom of the PIE.3 IU/mL) for 12 h at 4 °C. The oral epithelium was then removed and dispersed by pipetting 10 times and seeded onto Ti plates. OECs were cultured in defined keratinocyte serum free medium  and gentamycin (50 μg/mL) in a humidified atmosphere of 5% CO2 at 37 °C. All culture experimental data were analyzed 7 days after the cell seeding  were cultured as reported in the previous report . Briefly seeding .OECs were fixed in 4% formaldehyde for 10 min and permeated in 0.5% Triton X-100  for 3 min. OECs were then incubated overnight at 4 °C with a polyclonal rabbit anti-rat E-cadherin antibody  or polyclonal mouse anti-rat Integrin α3 (In-α3) antibody . Cells were further incubated with fluorescein isothiocyanate (FITC)-labeled secondary antibody  for 2 h at 37 °C. Actin filaments were visualized with tetramethylrhodamine -labeled phalloidin  for 1 h at 37 °C. Nuclear stain was done using 4′6-diamidino-2-phenylindole . Cells were observed using a fluorescence microscope .®  and immunoblotted with polyclonal antibodies , rabbit anti-rat In-β4 (1:100), or mouse anti-rat plectin (1:100)) for 24 h at 4 °C. Blots were then incubated with HRP-labeled goat anti-rabbit IgG secondary antibody  for an hour at room temperature. Pictures were taken using a chemiluminescence system (Amersham Biosciences).OECs seeded onto each substrate were lysed in lysis buffer and centrifuged at 13,000 g for 10 min. Proteins were separated on 7.5% polyacrylamide gel, transferred to immun-Blot PVDF membranesA cell proliferation kit  was employed to detect cell proliferation. Briefly, OECs were exposed to 5-bromo-2′-deoxyuridine (BrdU) in culture medium for 1 h and then fixed in 4% formaldehyde for 10 min. The presence of BrdU was visualized using mouse anti-BrdU monoclonal antibody (1:100) with FITC-labeled anti-mouse IgG .Scratch assays on Ti plates were performed as a model for wound closure . ConflueP < 0.05 were considered to be statistically significant.Data are expressed as means ± SD. One-way analysis of variance with Tukey’s post hoc test was performed. The survival rates of experimental implants for each time point were analyzed by the log-rank test. 2 peaks were observed. Unlike the result of Cont, both hydrothermal groups represented TiO2 peaks, as well as Ti peaks . For this purpose, a rat oral implantation model was used in the present study. This model has been used for more than ten years. Using these original models, the similarity between the junctional epithelium (JE) and PIE has been shown at the point of construction and function . AdditioAfter 16 weeks, we observed that the experimental implant models exhibited apical down-growth of the PIE. Furthermore, a marked positive area of Ln at all surfaces of the PIE was observed in the Ca-HT group and at the bottom of the PIE in the Cont and DW-HT groups. The distance of PIE migration on the Ca-HT implants was shorter than on the Cont and DW-HT implants, consistent with the in vitro data. Additionally, these results showed that the Cont and DW-HT implants might easily cause deep pockets, and have a high risk of leading to implant failure. We speculate that apical movement, as well as soft tissue integration between the peri-implant mucosa and experimental implant can be prevented by Ca-HT. Furthermore, limited expression of Ln in the Cont and DW-HT implants showed much weaker sealing of the peri-implant soft-tissue than the Ca-HT implants. The PIE with weak OEC adhesion to the Ti surface on the Cont implants seemed unstable as described above, and was more inclined to migrate. The Ca-HT group had a significantly larger number of OECs than the DW-HT group. In our previous studies, the presence of Ca on the Ti surface effectively contributed to greater attachment of OECs. This result is consistent with the view that an enhancement of the cell activity by divalent cations, such as Ca or Mg ions, promotes initial epithelial cell attachment to a negatively charged substrate . TherefoWound closure speed in the scratch assay is affected by both the migration of outermost cells and the proliferation of the following cells . In the 2 hydrothermal-treated Ti. Additionally, strong adhesion to the substrate is also one of the most important factors for cell proliferation . Here, w2 nanoparticles were reported to cause inflammation partly due to the change in several immune response relevant signaling pathways [2 surface is basically stable in body and plays a crucial role for the excellent biocompatibility of titanium [Recent literatures indicated that oral implants are distinguished as foreign bodies by immune system. As a result, oral implants evoke a mild immunoreaction. And implants survive in the body in a delicate positive immune balance based on the individual host immune system ,27. Titapathways , but pastitanium ,31. Howetitanium ,33. Besititanium . The resAs reported previously, Ca-HT enhanced the bone-titanium contact . SurfaceA lot of attempts have been reported to enhance soft tissue integration with abutment. For example, using abutment with smaller diameter than the diameter of the implant body, so-called platform-switching, is proved to prevent epithelial down-growth and foreign body penetration . This isThe limitation of this study is that we employed rat oral implantation model. Healing capability and the defense against infection of rat may be stronger than human. In contrast, both plaque control and the control of occlusal force were not provided. As a result, gingival reaction toward the implanted material in rat may be slightly different from human. Other limitation is that we used implants with machined surface because we would like to eliminate other factors than hydrothermal treatment. And we employed one-piece implant incorporating both the implant body and an integral fixed abutment in one piece. Both machine-surfaced implant body and one-piece implants are less popular in the clinical situation. Since rat oral cavity is very small, two-piece implant is difficult to use. But rat oral implantation model is well-established and using this model, we could reduce the use of larger animals.2 hydrothermal treatment can improve the adhesion of PIE cells to the Ti surface of titanium implants and may create a secure seal at the epithelium-implant interface and reducing epithelial down-growth, even after 16 weeks of implantation into rat maxillae. Insufficient adhesion of soft tissues can lead to the formation of deep peri-implant pockets, raising the likelihood of periodontal complications. In addition, this procedure enhanced the formation of surface oxide layer without altering surface topography. This approach may have a potential to enhance long-term implant stabilization. As a result, the null hypothesis “Ca-HT has no impact for the prevention of epithelial down-growth to maintain the structure using rat implant model for 16 week” was rejected within the limitations of the present study.The present data suggest that activated cell attachment by CaCl